This assumption is supported by a decreased level of the mutated MetAs observed in insoluble protein fraction under a temperature shift from 30° to 45°C compared with the native MetA protein (Additional file 4: Figure S3). If a native protein is thermodynamically unstable and/or functions under stress conditions, then kinetic stabilization could enhance the functional properties of the protein [21]. Furthermore, improved kinetic stability is tightly associated with protease resistance [22]. Notably, the MetA mutants were more resistant Selleckchem JNK inhibitor to proteases; in vitro reconstitution experiments confirmed the resistance of the MetA mutants to the

ATP-dependent cytosolic proteases, including Lon, ClpPX/PA and HslVU (Figure 6). Previously, the aggregated MetA protein was identified as a substrate for intracellular proteases Lon, ClpPX/PA and HslVU [6]. Biran et al.[6] assumed the combinatorial action of these proteases on

MetA degradation because the protein stabilization was detected in the triple deletion mutant lon, clpP, hslVU but not in any single (lon, clpP, hflB and hslVU) or double (lon–clpP) deletion mutants. Figure 6 In vitro degradation of the native MetA protein and stabilized I229Y mutant by the ATP-dependent proteases Lon, ClpP/X and HslVU. Degradation reactions were performed at 37°C with or without ATP as described in the Methods section. Untreated proteins indicate the positions of native MetA (the central lane of the upper gel) and mutant I229Y (the left lane of the lower gel). Densitometry results were normalized after setting the MetA selleck chemicals amount before ATP addition equal to 100%. The results are plotted as the mean and standard deviation of two independent experiments. Previous studies have

shown that the dnaK gene is not essential for growth and protein Selleck FK228 folding at 30°C but is required at temperatures above 37°C or below 15°C [23]. Here, we showed that the defective growth see more of a ΔdnaK mutant at 37°C can be partially restored using a stabilized MetA (Figure 4). This result suggests that the growth defect of the DnaK-deficient strain is primarily due to non-functional MetA because MetA, an inherently unstable protein even at the physiological temperature of 37°C, requires folding assistance from the DnaK chaperone system. The stabilized MetA mutants also partially restore the growth defects of protease-deficient strains at 42°C (Figure 4). We also examined whether the temperature-sensitive mutations (ΔmukB, ΔbamE and Δlpp) affecting other cellular processes are suppressed through methionine supplementation at higher temperatures. None of the mutants showed improved growth, indicating that proper methionine supply is a major issue in the growth defects of both a ∆dnaK and the triple protease mutants.